Project Summary/Abstract Group 2 innate lymphoid cells (ILC2s) are tissue-resident lymphocytes with emerging roles in tissue development, remodeling, and allergic disease. In the lung, ILC2s are key instigators of allergic asthma, yet little is known about where ILC2s reside and how stromal interactions shape ILC2 development and function. We have found that lung ILC2s reside in specific perivascular ?niches?, where they intimately associate with a mesenchymal cell subset that expresses the cytokines IL-33 and TSLP. The goal of this proposal is to rigorously investigate the role and cross-talk of these ?niche? mesenchymal cells on ILC2 developmental localization, function, and induction of type 2 immune responses. Based on our preliminary data, we propose that niche perivascular mesenchymal cells impact ILC2 localization and function via the production of known cytokine signals, including IL-33 and TSLP, as well as other novel pathways, providing a nidus for adaptive Th2 immune responses. During inflammation, ILC2s cooperate with Th2 tissue-resident memory cells to cross- regulate mesenchymal cell function and cytokine production. In Aim One, we will define how adult lung ILC2 ?niche-associated? mesenchymal cells impact ILC2 activation and induction of adaptive type 2 immune responses. Global deletion of niche mesenchymal cells impaired lung type 2 immune responses, and we predict that cytokines such as IL-33 and TSLP, as well as other unknown factors, contribute to the activation of ILC2 and the type 2 immune response. In Aim Two, we will characterize the stromal cells and signals that govern the postnatal, developing ILC2 niche. Our preliminary data indicate the earliest ILC2 interact with and require mesenchymal perivascular niche cells for optimal expansion. We will test the development of these perivascular niches and their contribution to ILC2 localization and function in perinatal life, and how allergic challenges in this period alter the developing niche. In Aim three, we will explore how ILC2 and tissue Th2 cells cross-regulate niche mesenchymal cells. We have found that both ILC2, Th2, and IL-33 expressing mesenchymal niche cells expand during helminth infection, and ILC2 are required for optimal expansion and IL-33 expression by mesenchymal niche cells. Here we will test how ILC2-derived cytokines impact mesenchymal niche function during both perinatal development and adult immune challenge. We expect that signals from ILC2 dynamically regulate niche mesenchymal cell function and IL-33 expression to cause long- lasting changes in tissue immune tone and predisposition towards subsequent allergic asthma. Together, we use novel 3D imaging techniques, RNA-sequencing, and transgenic tools to answer a critical and unknown question: how are ILC2 locally regulated at their lung niches? We anticipate these studies will have fundamental implications for the control of ILC2 and type 2 allergic immunity in allergic asthma, and yield insight into how ILC2 impact both beneficial and pathologic type 2 immune responses in the lung.